JPS60218071A - Solid-phase support composite body having chromogen responding function for detecting antibody, antigen, receptor or ligand - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to assay techniques for antibodies, antigens, receptors and ligands, and more particularly to improved assay techniques containing chromogen-responsive markers for the measurement and detection of antibodies, antigens or receptors. Concerning the same phase support samurai union.

Because of the sensitivity and specificity it can provide to diagnostic technicians.

Radioimmunoassay and radioreceptor assay are important tools in clinical diagnosis. However, radioassays not only require the materials necessary to perform radioisotope labeling, but also require complex equipment for radioactivity detection. Therefore, this method is limited to well-equipped laboratories in societies and countries that have the necessary space for the appropriate equipment required by the detection technique. As a result, in many therapeutic situations it is not possible to perform assays with the sensitivity that radiometry provides. In such a situation.

These include private practice offices, urgent care clinics, local hospitals and treatment centers, and similar situations. Furthermore, a quick and simple test at home is also not possible.

drug without the need for radioisotope handling and therefore without the need for radioactivity detection equipment.

Attempts that have been made to enable the measurement and detection of steroids, peptides, etc. include the development of non-isotopic immunoassays, such as enzyme immunoassays.

One example of such achievements in enzyme immunoassay technology is the emergence of home pregnancy tests that allow women to determine their own pregnancy using a relatively simple detection method. Prior art related to the development of pregnancy testing tests includes the following: U.S. Pat.
No. 4,016,250, 4. .

94.963, 3,862,302, 3,655,
838 and 3,654,090; Van We
"Immunoassay using antigen-enzyme combination" by Eman et al.

FEBS 1etters, 15, 232 (1971'
): 5axena et al. "High-yield isolation of luteinizing hormone-chorionic gonadotropin Lycefree from bovine corpus luteum".

The Journal of Biological
Chemistry, VoL258, Associate 5. 341
0-3158 (March 10, 1983); 5ax
[Urinary Luteinizing Hormone - Radioreceptor Assay for Human Chorionic Gonadotropin: Detection of Luteinizing Hormone Surge & Pregnancy J.

Fertility & 5terility, Vo
l, 28. A2 (February.

1977); 5axena et al. [Diagnosis and Management of Pregnancy by Radioreceptor Assay for Human Chorionic Gonadotropin J, American Journal o
f Obstet-rics and Gynecol
ogy, Vol. 131. 97-109 (1978
(May 1, 2017). Particularly different diagnostic devices for carrying out the above assays are described in two examples, for example US Pat. No. 63.57'9.306 and US Pat. The test methods disclosed in these prior art generally
By a method such as a binding reaction between and an antibody (immunoassay) or a binding reaction with a receptor (receptor assay). Techniques developed for pregnancy testing (radioassays and enzymatic assays) are equally applicable to the measurement of other molecules, including, for example, but not limited to, steroid molecules, protein molecules, hormones, etc. As those skilled in the art will appreciate, the broad applicability of such enzyme immunoassay techniques is recognized in the art.

Despite developments in this field in recent years.

Prior art tests have not yet been sensitive enough to detect levels of antibodies, antigens, or receptors as low as 9%, which is necessary to make enzyme immunoassay techniques more comparable to radioimmunoassay techniques. Additionally, the prior art tests that approach the sensitivity of radioassays require equipment and equipment. The subject of the invention is for assay techniques as described above.

It concerns a more sensitive chromogen-responsive complex.

Aspect A of the present invention resides in reagents for the measurement and detection of a component of an antibody-antigen reaction. Two forms of such reagents are within the scope of this invention. One form is that the antibody is chemically bound to an insoluble homogeneous matrix or support.

This is a combination of a carrier carrying multiple chromogen-responsive label molecules and an antigen. Another form of reagent is one in which the antigen is attached to an insoluble homologous matrix or support through chemical bonds.

This is a combination of a carrier carrying multiple chromogen-responsive label molecules and an antibody conjugate. A preferred chromogen-responsive label is an enzyme. In the case of pregnancy determination, the antigen is CG and the antibody is anti-β-hCGf-globulin.

Another aspect of the invention is a method for measuring one component of an antibody-antigen reaction or a +117 receptor-receptor binding molecule (ligand) reaction. This measurement method is an immunochemical assay using the above-mentioned novel reagent.

This method involves contacting a sample containing the component to be measured with a predetermined amount of the reagent for a time sufficient for an immunochemical reaction to occur, separating the insoluble substrate of the reagent from the test solution, and discharging the remaining test sample at °C. It consists of treating the solution with a chromogen responsive to said marker. For example, in a pregnancy test or in the body fluid being tested, the presence of the ACG hormone is indicated by an appropriate color or color change depending on the particular chromogen system used. Yet another aspect of the invention is reagents and methods for the determination of a receptor or a molecule capable of binding to that receptor. antigens and antibodies.

Although receptors and molecules that bind to these receptors are examples of conformationally and biologically specific reactants that can be measured by the present invention, the present invention also deals with the use of specific reactants other than these. It can also be applied to the detection of

Advantages of the present invention also include the development of reagents that bind larger amounts of each marker reagent molecule than previously possible. An increase in marker abundance also greatly improves the sensitivity of measuring low levels of substances being detected and/or measured. For example, in pregnancy tests, another advantage is that it allows determination of pregnancy much earlier than was possible with traditional enzyme immunoassays. Yet another advantage.

The methods and reagents of the present invention are highly reliable in measuring antigens, antibodies, or receptors. Yet another advantage is that reagents have been developed that are stable and thus can be pre-prepared and stored well over long periods of time and have good handling properties. Yet another advantage is that the assay with the new reagents is simple to perform, increasing its usability by unskilled users and individuals at home. Further advantages will become apparent to those skilled in the art from the following description.

Next, the present invention will be explained in more detail based on the drawings.

The present invention provides reagents and methods for diagnostic tests that do not use conventional laboratory equipment, such as spectrometers or spectrophotometers, but instead use indicators that can be observed with the naked eye. The diagnostic test developed in this invention allows for home use by unskilled personnel. Furthermore, two diagnostic reagents of the present invention include:
Ideal for use in non-laboratory medical environments 9 such as a doctor's office, outpatient clinic or emergency room. A particularly accurate, sensitive and reliable assay method in the above environment.

Macroscopic indicators 2 that can be implemented quickly and relatively cheaply
For example, due to the use of hue-changing two-color straddle patterning, it is no longer necessary that trained laboratory technicians perform and evaluate the results of diagnostic tests. Despite such qualitative nature of the diagnostic reagents, quantification of the results is also easily ascertained, as described in more detail below. The novel reagent of the present invention enables the measurement of one component of an antibody-antigen reaction or a receptor-receptor binding molecule reaction, and is composed of five basic components.

These components include solid support matrices, antibodies (
or receptor), antigen (or receptor-binding molecule)
, carrier molecules, and chromogen-responsive marker or tracer molecules.

Of particular importance is the formation of single or complex reagents, which immediately distinguishes it from the prior art.

Although a wide variety of reaction techniques can be used to form the reagents of the present invention, certain novel and preferred reaction methods are now described. In particular, the use of human chorionic gonadotropin (ACG) in this method and its application in pregnancy diagnosis is exemplified. However, this special example is an illustration for explanation and does not limit the invention to this scope.

First, regarding the solid phase or solid support matrix,
Preferably, the matrix comprises a solid that can be treated to covalently attach antibodies, receptors or antigens thereon. A preferred matrix is nylon-6. However, other suitable conventional materials such as nylon, polybutadiene, polystyrene, latex, etc.
frisien-styrene coholimer, crow, 7i
o-su.

Support matrices such as cross-linked dextran, cellulose, liposomes (phospholipid bilayer membranes), F paper, etc. can be manufactured in various shapes, for example, rod-shaped.

It may be in the shape of a plate, a ring, two discs, a tube, a bead, two cones, one grain, etc.

To make the surface area of the support as large as possible.

Preferred to provide maximum concentration of reagents. Although physical adsorption techniques for attachment of antibodies or other reagents to the solid support may also be possible in some applications, it is also preferred to attach the solid support onto which the antibody, receptor or antigen is attached. processed to form a covalent bond. If you fall down, nylon-6
The solid support is treated with 9 e.g.
AHTL) to convert the amine group into a thiol group. The use of AHTL as a spacer molecule between a solid matrix and an antibody reduces the steric hindrance of the thiol-reactive groups and the antibody binding site, for example. This thiolated carrier can then be coupled, for example, to a maleimidized antibody (eg, anti-ACG). 7 or less, H
The amino group is non-reactive with the thiol group, so crosslinking is prevented in this way. Other methods for covalent attachment of antibodies or antigens to solid phases are described by Kawo.
[Improved conjugation method of peroxidase and protein Tsukuda J]
, Fed Proc, , 32 Abstract
840 (1973);
+ Psy-choneuroendocrinol
ogy'in Reproduction.

Edited by Zichella et al., Elsevier/Nbrth
Holand (1979); Po5t et al.
CIjnEndocrino1. Metab,, 5
0.169 (1980). These allow antibodies, receptors,
The antigen may be covalently attached to the solid phase by conventional means as necessary, desired or convenient.

As mentioned above, the second component of the reagent of the invention is an antibody, lysefree or antigen, which is treated (denatured) for reaction to the same phase. For the purposes of the present invention, antibodies broadly refer to monoclonal antibodies, monoclonal antibodies, monoclonal antibodies,
A mixture of two or more monoclonal antibodies in predetermined ratios, each having different binding and/or cross-reactivity to the same antigen, or each having different binding and/or cross-reactivity to the same antigen. It has a broad meaning that includes a mixture of one or more monoclonal antibodies and one or more polyclonal antibodies having the following. Such antibody mixtures may contain antibodies against the same antigen (e.g. AC
G) but probably act on different antigenic determining sites (epitopes) of the same antigen. Of course, this synergy was not fully understood before the advent of monoclonal antibodies. A wide range of receptors suitable for use in the immunodiagnostic tests of the invention should be understood based on the foregoing description.

Moyle et al. ``Use of monoclonal antibodies against subunits of human chorionic gonadotropin for testing hormone orientation and complexes with its receptors J, Proc, Nat 1. Ac.
ad, Sci.

USA, Vol. 79, 2245-2249 (198
April 2016); Ehrlich et al. [Achieving improved affinity for antigen by mixing two monoclonal antibodies J
, the Journal of Itrmunolog
y, Vol, 128.46 (June 1982);
Ehrlich et al., “Immunoassay: Trend Sensitivity Test with Mixed Monoclonal Antibodies J, 5ci-ence”
, Vol. 1.221, 279-281 (July 15, 1983). In this regard, the term "antigen" should be understood to include not only the antigen in an antigen-antibody pair, but also the second antibody in an antibody-antibody pair in which the second antibody is raised against the first antibody. be. That is, the first antibody (eg, anti-r-globulin) is also an antigen targeted by the present invention. Such antibody-antibody pairs.

For example, they are used when antigens are difficult to obtain and/or when supplies are difficult and/or expensive. It is within the scope of the present invention to prepare reagents based on the antibody-antibody pairs described above in accordance with the teachings of the present invention.

For pregnancy tests it is currently preferred to couple the antibody to a nylon or other solid matrix, although it is of course also suitable to couple the antigen or receptor to a solid phase. Antigen-antigen reaction and IJ receptor
Regardless of which components of the susceptor-binding molecule reaction are bound in the same phase, 9 they are bound to the labeled conjugate for the preparation of the reagents of the present invention. The antibody-antibody bond (or coupling) is not a covalent bond, but a Panderbarska type that can be replaced by the component to be measured.

Therefore, the remaining three components are preferably formed into a conjugate, and the conjugate is combined with the treated solid matrix.
Add IJ Lux. Conjugation is the detectable binding of protein or non-protein substances to other protein or non-protein substances. In the reagents of the invention, the ideal conjugate consists of one or two molecules of antigen, lysefree or antibody (as determined by the components bound in phase) and a number of chromogen-responsive markers or Kuhatracer molecules. For example, conventional reagents have been covalently linked directly to antibodies or antigens and enzymes, providing a maximum of only 3 or 4 enzymes per antigen or antigen molecule. It is clear that the amount of enzyme present determines the sensitivity of the assay to be able to detect components of interest that are present at low levels. This shows that the reagent of the present invention has the ability to determine pregnancy (with the naked eye) without delaying the expected start of menstruation. If the zygote of the present invention is used.

Broadly, for example, about 1 to 15 molecules of enzyme are carried in the conjugate per molecule of ACG antigen in the conjugate, typically about 5 to 10 molecules of enzyme per molecule of antigen. The pantheon of enzymes useful as tracers or markers also include horseradish peroxidase, alkaline phosphatase, catalase, and the like. Enzymes are preferred as markers. This is because enzymes can be easily detected even in very small amounts. However, other markers such as phenols, enzyme cofactors.

Dyes (e.g. azo dyes), chemical fluorophore precursors.

Enzyme substrates, fluorescent dyes, fluorogens, fluorescent quenchers, heme, and the like can also be used as markers of conjugate formation. For example, 5chall and Tenoso [Alternatives to Radioimmunoassay 2 Labeling Agents and Methods].

Cl1n, Chem, Vol, 27. A 7
(1981).

The requirements for a suitable marker for use in zygotes are 9.
It reacts with chromogens and its presence can only be detected with the naked eye.

The carrier molecule of the conjugate must be soluble in the reaction solvent (typically an aqueous solvent), and this requirement practically limits the molecular weight of the carrier. Furthermore, phases that are too thick, with two molecular weights, exhibit retarded reaction rates, making their use impractical. Preferred carriers often do not substantially exceed a molecular weight of about 70,000, such as sea-close polymers (e.g., Ficoll®, bovine serum albumin (BSA)) and similar known carriers. It is a molecule. A variety of conjugation techniques with reaction specificity have been proposed in the field9 and can be found, for example, in the following literature:
Engvall et al., “Enzyme-linked immunosorbent assay (ELISA): Assaying the determination of immunoglobulin G, J, Irrmunochemistry, 8.87
1 (1971) s Kawo et al. ``Improved conjugation method between peroxidase and protein'' (mentioned above); 5axe
na et al. “Development of a centrifugation-free solid-phase enzyme assay for LH for ovulation testing” (mentioned above); Carlsson et al. “
Protein thiolation and reversible protein-white matter junction [
Biochem J, .

173.723 (1978); Khan et al. [Use of purified gonadotropin receptors in the development of enzyme receptor assays (EA) for luteinizing hormone and human chorionic gonadotropin J, Enzyme L.
abelled immunoassay of HP
Rmpnesand Drugs, S, B, P
a1 edition de Gruyler & Co.

(1978); Lee et al.
Preparation of COOH peptide-carrier conjugates J, Mole
-cular Irrmunplogy, 17.74
9-756 (1980).

FIG. 1 schematically explains the reagent of the present invention for an example of measuring hCG in the urine of a pregnant woman.

This figure is for illustrative purposes only and is provided to facilitate visual understanding of the detailed description of the present invention. In FIG. 1, the solid matrix is composed of AC attached to carrier molecules.
It consists of an antibody coupled to a G molecule. It supports somewhat complex molecular structures. The carrier molecule is not bound to the antibody40
It also has 0 molecules as well as several molecules of marker, which in FIG. 1 is horseradish peroxidase. The illustration of this reagent according to FIG. 1 illustrates various aspects of the invention.

First, the carrier molecules typically carry one or two molecules of antigen such as ACG per molecule. Having more than one molecule of 800 per carrier molecule increases the 2 reaction rate and promotes the binding reaction of the phase molecule to the antibody bound to the +7x. Secondly.

The ratio of HRP marker molecules per AC0 molecule is 6:
1. This ratio is within the wide range of 4-15:1 mentioned above. The important point is.

According to the specific structure of the reagent shown in FIG. 1, each conjugate molecule composed of the present reagent provides an HRPV of 91 molecules per 2/2 ACG molecule. The sensitivity of the reagent is increased by the high ratio of markers per molecule of antigen to be measured. of course.

The matrix supports an infinite number of the molecules shown.

Only one of them is shown in FIG. 1 for clarity.

Another important point is that the bond between the antibody and the matrix is a covalent bond that is difficult to cleave under the typical reaction conditions in which the reagent is used. For example, the ACG to be measured in urine at the starting point is indicated by the triangular symbol ACG, and the two points m&
! This shows the # exchange reaction that occurs when the matrix comes into contact with the urine of a pregnant woman. The ACG molecule to be measured is present in liquid urine.

Reagents, on the other hand, are in solid form.

In FIG. 2, the state after the result of the ct conversion reaction is shown.

A solid matrix with covalently bound antibodies can be used for measurement from pregnant women's urine! The hCG molecule is conjugated to the antibody and held at °C. This solid matrix is removed from the remaining liquid reactants. After removing the matrix, carrier molecules containing HRP marker molecules are present in the residual liquid. It is this carrier molecule substituted with the ACG molecule to be measured that is visually assayed by adding appropriate chemicals to detect the presence of the HRP molecule. The resulting carrier molecule has an equivalent number of HRP marker molecules per molecule of ACG displaced from the matrix. Therefore, the sensitivity of the present invention can be obtained.

Two representative general reaction schemes for conjugate formation are briefly described below and detailed in the Examples below. Regardless of which method.

Antibodies, antigens, or receptor and chromogen-responsive markers are denatured to react with or bind to carrier molecules. In their denatured state, no binding or coupling occurs between the marker and the antibody, antigen or receptor. It should be noted here again that such a reaction scheme is preferred for illustrative purposes, but is not intended to limit the invention. The first type of reaction is what can be called the formation of a "sandwich", in which enzymes or other markers are stacked in one continuous layer.

Increase the amount of marker per molecule of carrier. It should be noted that the ability to increase the number of moles of enzyme or marker on many carriers is more limited by steric hindrance than by the number of two reaction sites. Layering or sandwich technology is one way to overcome these limitations. Basically, this reaction method! Two methods can be considered to facilitate implementation. One method is to maleimide the carrier molecule.

and thiolation of the marker and antigen, and simultaneous or subsequent reaction of these components in the desired ratio. This method tends to create difficulties in purification without crosslinking.

Another method is to thiolate the carrier, maleimidize the antigen and marker, and then react them. This method allows easy purification without crosslinking. Preferably, for example, it can be implemented as follows. BSA or other carrier is thiolated using N-cetylhomofuffhinethiolactone (hereinafter A, HTL). Horseradish peroxidase (H
(hereinafter referred to as RP) or the other two purple 6-maleimidocaprone ffN-hydroxysuccinimide ester (hereinafter referred to as MC
(abbreviated as 8)) to maleimide. Thiolated BSA
&jIt may be reacted with up to 4 to 5 moles of maleimidized HRP.

This reaction product can then be reacted with a dithiol, such as 1,6-dithiohexane or dithiothreitol, and further maleimidized HRP can be added. This addition may be repeated as many times as necessary to obtain additional molar amounts of enzyme marker per mole of BSA carrier. Next, maleimide ACG (by MC8) was added to HR8
carrier molecules sandwiched with and thiolated BSA
The reaction is carried out at a ratio of about 1 to 2 moles of maleimidated ACG per mole. The resulting conjugate can be coupled to antibody-treated nylon or other homologous phase to form the novel reagents of the invention.

The above “sandwich” using a controlled reaction
Variations on the method also include the formation of a compound of acetic acid (or other marker). Although such polymerization is a non-specific reaction using a cross-linking reaction.

This crosslinking reaction method is excluded in the prior art listed herein. Enzyme crosslink formation is carried out by carbodiimide,
geltaraldehyde, polyisocyanate) (mul
ti-ispcyanates), and similar divalent and multivalent reagents that can form enzyme-enriched polymers. This polymer was then used to form conjugates as described herein. A variety of specific reactions for the formation of antigens, antibodies or receptors enriched with enzymes or other markers can be used.

The second general reaction scheme can be carried out in two ways.

This method is based on N-succiniidyl-3-(2-pyridylthio) reported in Carlsson et al., “Protein thiolation and reversible protein-protein conjugation” (cited above).
- Using propionate (hereinafter abbreviated as 5PDP).

This reaction mode can also be carried out, for example, by using Ficoll 7O as a carrier molecule and reacting it with about 5 to 20 moles of 5PDS per mole of Ficoll 70.

hCG or other antigens may also be reacted with 5PDP.

One or two thiol groups can be present per mole of AC0. 5PDP-modified Ficoll 7o and 5PDP-modified hCG can then be reacted in a ratio such that the Ficoll molecules have 1 or 2 ACG molecules per carrier molecule.

HRP or other markers are then added to 5PDP or A
, HTL'& can be thiolated at °C. It is then reacted with a thiolated HRPVACG-Ficoll reactant to yield a conjugate having 1 or 2 molecules of ACG per molecule of Ficoll and about 4 to 15 molecules of HRP per molecule of Ficoll.

ACG, HR
By separate thiolation of P and Ficolla θ and subsequent simultaneous reaction of these three reactants. In this case as well, the molar ratios of the three components are the same as in the first method.

Regardless of the method adopted.

The resulting conjugate is then coupled to antibody-treated nylon or other solid support to form the reagent of the invention. The reagents thus obtained are stable, can be preformed for ease of handling, are stable and can be stored for long periods of time. Also, 9 the reagents of the present invention do not bind to free amines.

To bind the conjugate to antibody-treated plates.

The reactants may be contacted for typically about 2 hours to 5 days, more frequently about 12 hours, depending on the temperature. The resulting rigidly bound complex is then briefly washed to remove the conjugate that is attached by surface tension. However, in order to prevent dissipation of the bonded material,
It is better not to immerse the solid phase in water. Since the aqueous prepared conjugate is easily dissolved in water, it should be noted that the contact time of the solid-supported conjugate with water should be as short as possible. Finally, to inhibit dehydration of the complex on the surface of the nine-sided phase, a protective coating of propylene glycol-ethylene glycol, mannitol or similar substances is applied to the solid phase by dipping or other conventional methods.

The resulting solid phase supporting the reagents is then air dried and ready for use.

Although specific reagents have been used as examples in the above description, this is for illustrative purposes only; many of the reagent components described above are interchangeable with others, and thus other reagent components may also be used. Additionally, although much of the description of the 9 reaction has been based on pregnancy testing, 9 the reagents of the present invention may have other specific uses as well. For example, palpation of the uterus or sonography (sonography).
If no fetal implantation is observed by graphy (graph), quantification of ACG is performed in mW to confirm the presence of an ectopic pregnancy. Quantification is performed using pre-existing data for dilutions of known concentrations. The very low level of ACG production associated with ectopic pregnancy can be easily detected using the reagent of the present invention, as it is highly sensitive due to the use of a large amount of enzyme or other marker per molecule of the reagent. . Other uses of the reagents of the invention include the determination of molecules such as steroids, protein molecules and hormones. In this regard, 1 the reagents of the present invention can be used to detect female nistrogiens, proshosterone and luteinizing hormones, using body fluids such as urine and blood.
・By measuring, it can be used for ovulation and infertility tests. Again, the sensitivity of the reagents of the invention.

Example L&F - even allows the use of saliva, which has been reported to be used to detect prodiesterone and estrozaene. Other uses include the use of this reagent in pregnant women who have experienced miscarriage. The attending physician or the pregnant woman herself can easily monitor the ratio of l) oar.

Decreased estriol levels9 indicate a pregnancy disorder, such as a malfunctioning or not functioning placenta.

In order to explain other specific uses of the reagent of the present invention, an ovulation test conventionally performed by LH detection as described above will be described. The sensitivity and adaptability of the reagents of the invention are
Varnishes for predicting and testing ovulation (precise testing)
Allows measurement of both p-diene and LH. For example, the reagent of the present invention.

Alkaline phosphatase was used as an enzyme marker for nistrodiene, and HRP was used as an enzyme marker for LH.
V may be used. Of course, mixtures of nistrogienes and LH and their corresponding antibodies can then be used for the preparation of the reagents according to the invention. In this case, the assay reaction is 2.For example, indoquine phosphate is used as a chromogen for nistrodiene (blue coloration indicates a positive result) 9. Then amine antipyrine is used as a chromogen for LH (red coloration indicates a positive result) can be used continuously. As a result, if there is a purple color, Nistrodiene and L
H suggests the existence of both. Advantages of such dual detection methods include the generation of characteristic colors (and/or bacoons) and the use of one or more chromogen-responsive markers.

The advantage is that chromogens can be used that alone produce colors that are weak or difficult to detect. Furthermore, the risk of false positives due to having to measure two or more hormones (or other molecules) is reduced. Therefore, in the reagent of the present invention 1
The use of mixtures and combinations of the various components is within the scope of the invention.

The implementation of the present invention will be described based on the following examples, but is not limited to these examples only.

All references herein are incorporated by reference.

Example 1 Preparation of anti-ACG-labeled nylon 6 matrix
1 m) was immersed in a 3 N HCl solution for 1 hour at 37"C. The plate was then thoroughly washed with running water and then rinsed three times with distilled water. The aminated plate was soaked in a 1 molar solution of imidazole saturated with element 9. 0.05 mol AHTL in
The mixture was thiolated overnight. Wash with 0.2M acetic acid.

Elmens reaction (Elmens reaction) is carried out in the washing solution.
on) until no thiol compounds were detected.

Anti-β-ACG prepared and purified by conventional methods was lightly maleimidized with MC8 (about 1 to 2 maleimide groups per mole of r-globulin), purified by gel filtration, and lyophilized.

Thiolated nylon 6 plates containing 0.04% EDTA and 0.2 μ/ml maleimide r-globulin;
01 molar succinate buffer saturated with element 9 (pH 6,
0). Dip plates were photographed in this solution overnight at room temperature. The plate was then immersed in 15% saline containing N-ethylmaleimide to block free SH groups remaining on its surface. After immersing for 30 minutes, the plate was taken out and washed eight times with 15% saline, and then thoroughly washed with water as many times as necessary until the point at which the washing solution no longer showed absorption at 280 nm was reached.

The antigen binding capacity of the plate is 1 with known specificity and activity.
21) Measured by radioimmunoassay using IACG.

Example 2 The amine groups on the surface of the ACG molecule were sufficiently maleimidized with MC8, and purified by gel permeation chromatography. HRP
Lightly thiolate the enzyme with AHTI.

'The HRP contained about 1 to 2 thiol groups per molecule [to monitor the progress of the thiolation reaction].

Fluoroscamine-fluorometric method (Fl-uo
roscamin-Fluorometric met
hod)]. The thiolating enzyme product was dialyzed against 0.2 molar acetic acid and lyophilized.

Both the thiolated hCG product and the thiolated HRP enzyme were dissolved in 0.1 molar ψ buffer (pH 6,0) containing 0.04% EDTA and saturated with 9. The obtained conjugate was purified using a Sephacryl S-200 column.

Preparation of HRP-BSA copolymer About half of the amino groups of BSA were thiolated with AHTL.

The thiolated BSA was dialyzed against 0.2M acetic acid and lyophilized. Each HRP enzyme molecule was then maleimidized by HRPIMC8 so that it had about 3 maleimide groups. These denatured proteins were then washed with N-saturated 0.1 M succinate buffer, pH 6.0, O 14% EDTA.
, about 4-5 HR per BSA molecule.
BSA with P molecules was produced. The polymerized product was separated from the monomeric reactants by conventional gel filtration chromatography due to its increased molecular weight.

The separated BSA-HRP copolymer was reacted with 1,6-dithiohexane (or dithiothritol) to
One to two thiol groups were introduced into each (R, P) attached to the SA molecule. This thiolated product was reacted with an additional portion of the maleimidized HRP prepared above. The resulting product The product was separated from monomer HRP by gel filtration and lyophilized.

This process can be repeated as many times as necessary to obtain the multilayer HRP-BSA product V.

The ACG molecule was lightly maleimidated with MC8 so that 1 to 2 equivalents of maleimide groups were present per mole of ACG. The multilayer HRP-BSA product was then reacted with dithiothreitol (or 1,6-dithiohexane) to introduce 18H groups onto the last layer of HRP molecules. Maleimidated hCG and thiolated? , :HRP-B
The SA product (1:10 molar ratio of each acid) was then treated with N2-
Saturated 0.1 M succinate, pH 6,0,0,04%
Dissolved in EDTA and separated the product by Kegel filtration. The proportion of HRP and ACG in the enzyme-labeled hormone conjugate product.

Spectrophotometry for I (RP)
ate buffer, pH 7.0, mixed with dilute hydrogen peroxide) and.

Measured by RIA for ACG.

Some of the r-globulin-labeled nylon plates were prepared separately as described above, containing each ACG7BSA-HRP conjugate.
2% BSA-PBS solution (approx.
The antigen binding sites were saturated by immersion in a conjugate with a titer of 7 IU for 5 days.

Example 3 HRP-Ficoll-ACG Method A 021M borate buffer, p) T8.5. amine hexyl-phyco-/”70'42 dissolved in the minimum amount of
0 molar excess of N-succinimidyl-3-(2-pyridylthio)-propione-) (5P dissolved in DMF)
DP) for 45 minutes at room temperature. The thiolated Ficoll 7o product was purified by gel filtration on a Sepha, Dex G25 aqueous column, and the material recovered from the column void was lyophilized. The extent of 5PDP denaturation was determined by spectrophotometry when 2 was released from Ficoll 7o by reacting with thiol compounds 2, such as cysteine.
-Molecular extinction coefficient of thiopyridone 8.0O8X103M-
The optimum number of 5PDP groups was determined using 1(').

The thiol derivative of ACG was added to the thiolating reagent as described above for the thiolation of HRP.
1 was prepared using P. The extent of thiolation was followed by consumption of amine groups in the carrier molecule and monitored by fluorescence. What is the optimal degree of thiolation?

There are 18 HI equivalents per ACGI equivalent. thiolated hC
G product v was subjected to gel filtration on Sephadex G-25 using a 0.2 M acetic acid solution. The purified thiolated ACG'/r recovered from the void section of the column was analyzed spectrophotometrically for recovery and -Hm content. The product was brought to pH 4.7 by addition of sodium bicarbonate under nitrogen atmosphere.

It was used for coupling with the prepared thiolated Ficoll.

Sufficient amount of lyophilized 5PDP-Ficoll was added to the pH-adjusted thiolated hCG solution to adjust the respective molar ratio to l:
It was set as 2. The reaction was carried out at room temperature in a nitrogen atmosphere, and the progress of the two reactions was monitored by spectrophotometry (0, D).

343 nm).

0, D, 343 nm when the maximum value was reached.2 The reaction mixture was ultrafiltered through a PMIO membrane to approximately 25 rrQ/m
Concentrated to l.

The ami7 group of HRP dissolved in 1M sodium bicarbonate is citraconylated and oxidized by the addition of sodium periodate to remove a portion of its carbohydrate to yield an aldehyde group, which is then converted to b6-diamid. It was subjected to radical amination with a 2M solution in hexane. The product was then desalted and lyophilized. Aminated HRP
HTL thiolated to the extent that only one mole of AHTL was received for each mole of HRP-NH2. The thiolated HRP was purified by gel filtration on a 0.01 acetic acid-Sephadex G-25 column. Collect the material in the void area and powder it with sodium bicarbonate in an N2 atmosphere. The pH was adjusted to 4.7.

Finally, the thiolated HRR solution was mixed with the concentrated ACG-Ficoll conjugate solution (pH 4,7) and the mixture vN
The mixture was shaken at room temperature under 2 atmospheres for 16 hours.

The molar ratio of (hca:ficoll:)IRP is 2:1:(
4-15)). reaction? Add enough cysteine to stop the remaining free 5P on the Ficoll-furo molecule.
The DP group was converted to a non-reactive S-cysteinyl-8-propionyl adduct. The product was purified by gel permeation chromatography through a Sephacryl 200 column in 0.2 M ammonium sulfate, 0.05 M sodium phosphate + I)H6,8 to remove unconjugated ACG, HRP and cysteine. The void material was collected from the column and analyzed for ACG content by RIA and HRP content by spectrophotometry. product'&
0.05% Tween 20-0.1M sodium phosphate,
It was diluted to approximately 1.0 IU/ml at pH 6.8.

Amino-HRP and ACG molecules were each separately thiolated with 5PDP as described above, and the resulting thiolated derivatives were purified and lyophilized. Thiolate with amino-hexyl-ficoll 7o reagent AHTL.The appropriate amount of thiolation is amine-hexyl-ficoll 7.
0 to 1 mole), the HE composition is 0
0.1 molar acetic acid at 1-4'C - Sephadex G-25
It was purified by gel filtration through a column. Collect the material in the void area and remove the acidity using powdered sodium bicarbonate.
Adjusted to H4.7. Lyophilized HCG and HRP
of each 5PDP derivative was added to a dilute solution of thiolated "Ficoll 7o" after gel filtration. The reaction was carried out at room temperature with A
Optimum molar ratio of CG: Ficoll 70: HRP 2:1:(
4-15). 9 at the end of this conjugation reaction
A sufficient amount of S(2-pyridylthiol) cysteine was added to the reaction mixture to convert the remaining thiol groups on Ficoll 7o to non-crosslinkable side chains. The obtained product was reduced to 0
．． 2 molar ammonium sulfate - 005 molar sodium phosphate + I) H6,8, Sephacryl S -200 in solution
It was purified by gel filtration through a column.

Example 4 A conjugate of ACG, HRP and Ficoll 7o was prepared by the method of Example 3, and this conjugate was bonded to the r-globulin sensitized nylon plate obtained by the method of Example 1. 0.5 mi of non-pregnant urine was added to 5 rectangular test vessels. /I62 of the container. 3. To the urine added in 4 and 5, 12,525, 50 and 100 mIU of ACG (second international standard) per ml of urine were also added.

The conjugate-bonded nylon plates were simultaneously inserted into each container and left at room temperature for 30 minutes. Each plate was then removed. Then 0.5 ml of substrate solution was added to each container. The substrate solution was 005 in 1.6% phenol aqueous solution.
% Aminoantipyrine'& 0.2M K 2 HP
04 Hara 77 + pH7-0+NakanoQ, 03%H
, 02 solution at a ratio of 1:1.

After 10 minutes, the container A l l'lj showed no change in the color of the test urine. Containers /162 and /163 had a deep pink color, and container scraps 4 and 5 had a dark reddish-purple color.

These results show that the nine assay methods yielded 12.5 mIU in urine.
/m/! This shows that a relatively small amount of hCG can be detected, and ACG is not detected in the urine of non-pregnant women.

[Brief explanation of drawings]

FIG. 1 shows a schematic diagram of the reagent of the present invention, and also shows the exchange reaction pattern when measuring ACG in urine of pregnant women, for example. FIG. 2 represents a schematic representation of the exchange products resulting from the reaction measured in diagram H1. Each symbol in the figure has the following meaning. -Antibody 1 For example r-globulin ＼ Antigen of O reagent 9 For example ACG CII) Carrier molecule 9 For example Ficoll 7 ◇ Marker, for example enzyme like HRP △ Antigen to be measured 9 For example CG in pregnant woman's urine (No change in content) Procedural amendment 2nd day of Showa 'go' Yuzuki 2nd Showa 1999 Patent Application No. 12/ψ1 Relationship to Case Patent Applicant Address %11 ri01shi"'<Z・lx・incoho Itte 21μ゛4, Substitute person 5, Subject of correction/

Claims (1)

[Scope of Claims] (il (reaction product of a solid support matrix bound with an al antibody and a conjugate (hereinafter referred to as a conjugate) of an antigen, a carrier molecule, and a chromogen-responsive marker; (bl a homogeneous support matrix bound to an antigen and an antibody;
Reaction product of a conjugate of a carrier molecule and a chromogen-responsive marker; (reaction product of a conjugate of a receptor-binding molecule, a carrier molecule, and a chromogen-responsive marker with an in-phase support matrix bound to a cl receptor; a reaction product selected from the group consisting of: A reagent for measuring one reaction component of an antibody-antigen reaction or a receptor-receptor binding molecule reaction. (2) The reaction product is (at or (bIO). A reagent according to claim 1. (3) A reagent according to claim 1, wherein a mixture of the components is used for the preparation of the reagent. (4) The matrix is nylon, polybutadiene. Folystyrene, butadiene, -styrene copolymer. (5) The reagent according to claim 1, which is selected from the group consisting of glass beads, agarose, cellulose, liposomes that are phospholipid bilayer membranes, latex, dextran, and filter paper; (5) the matrix is nylon; (6) The reagent according to claim 2, wherein the antigen is a gonadotropin hormone. (7) Marker: enzyme, phenol, heme, enzyme cofactor, dye, chemiluminescence. substance anterior body, enzyme substrate,
A reagent according to claim 1 selected from the group consisting of fluorescent dyes, fluorescent ends, fluorescent quenchers and mixtures thereof. (8) The reagent according to claim 7, wherein the marker is an enzyme. (9) The reagent according to claim 8, wherein the enzyme is peroxidase. 2. The reagent of claim 1, wherein the carrier molecule of the ad conjugate is a searose polymer, bovine serum albumin, or a mixture thereof. The reagent according to claim 10, wherein the carrier molecule of the a■ conjugate comprises a Shakrose polymer. 3. The reagent of claim 2, wherein the t21 conjugate contains about 1 to 15 molecules of marker per molecule of antigen or antibody. 3. The reagent of claim 2, wherein the aj conjugate contains about 4 to 15 molecules of enzyme per molecule of antigen or antibody. a4 reaction product (a) is reacted with a triad consisting of a sucrose polymer carrier having 1 or 2 molecules of human chorionic gonadotropin and about 4 to 15 molecules of berooxidase per sucrose polymer molecule; The reagent according to claim 1, comprising a nylon-6 support matrix 7 to which anti-β-human chorionic gonadotropin is bound. (a) Solid phase support M) Covalently bond IJ wax antibody. (bl) An antibody-
A method for producing a reagent useful for measuring one component of an antigen reaction. 16) A method according to claim 15, in which a mixture of components is used for the preparation of the reagent. 16. The method of claim 15, wherein the in-phase support matrix is made of nylon. 16. The method according to claim 15, wherein the reaction is carried out by using the t8 antigen and the antibody in reverse. No. 9: The method according to claim 15, wherein the antigen is a gonadotropin hormone. 16. The method of claim 15, wherein (c) the chromogen-responsive marker is selected from the group consisting of enzymes, phenols, heme, and mixtures thereof. (21) The method according to claim 4, wherein the marker is an enzyme. (22) The method according to claim 1, wherein the enzyme is peroxidase. (23) The method of claim 15, wherein the conjugate has 1 or 2 molecules of antigen and about 4 to 15 molecules of marker per carrier molecule. (24) The solid support matrix is nylon. 24. The method of claim 23, wherein the carrier molecule is bovine serum albumin or a sucrose polymer, the marker is an enzyme, and the antigen is human chorionic gonadotropin. (25) (a) Providing a reagent that is a reaction product of a homogeneous support matrix bound to an antibody and a conjugate of a carrier molecule of an antigen and a chromogen-responsive marker. (b) A sample containing the component to be measured. Contact with the reagent for a sufficient time to generate an immune response. (C) removing the support matrix from contact with the sample; (d) adding a chromogen responsive to the marker to the sample; and (fe) examining the sample to determine whether a color indicative of the detection threshold of the component to be measured occurs in the sample; 1. A method for immunologically measuring a component of an antigen-antibody reaction. (26) The in-phase support matrix is nylon. 24. The method according to claim 23, wherein the marker is an enzyme and the antigen is a gonadotropin hormone. (27) The method according to claim 24, wherein the hormone is human chorionic gonadotropin. (28) The method according to claim 25, wherein the enzyme is peroxidase. (29) The method according to claim 25, wherein the carrier molecule of the conjugate is a sea-close polymer. (30) The method according to claim 25, wherein a mixture of components is used for producing the reagent. (31) The method according to claim 30, wherein the step (di) uses a mixture of chromogens. (32) The method according to claim 30, wherein the step (di) is continuously repeated each time with a different chromogen. The method described in Section 3. (33) The test sample of (a) is a mixture of nistrogien and LI-() formed from their respective antibodies and a chromogen-responsive marker, and in step (dl) a mixture of multiple chromogens is formed. (34) The marker consists of alkaline phosphatase,
and step (dl is p-nitrophenol and endoxyl phosphate)
-1e) in any order, the method according to claim 33 of the present claim. (35) Measurement of one component of an antibody-antigen reaction or a receptor-ligand reaction in which one component is an insolubilized form and the other component is bound to an enzyme; A reagent characterized in that it is supplied in the form of a conjugate of a component and a carrier molecule having about 4 to 15 molecules of a chromogen-responsive marker per molecule of the component.(36) The reagent according to claim 35, wherein the chromogen-responsive marker is an enzyme. (37) The reagent according to claim 35, wherein the first component is an antibody or an antibody center, and the second component is an antigen or a ligand. Reagent according to item 35.